biology essay

Bacillus Thuringiensis Scientific Classification Biology Essay

Published: 23, March 2015

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Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterial species of genome size of 2.4 to 5.7 million base pairs which during its sporulation produces insecticidal parasporal crystal (cry) proteins used as highly specific insecticides in agriculture and forestry because they are specifically toxic to particular orders and species of insects, like Lepidoptera, Diptera, and Coleoptera.

These genes are also used to engineer insect-resistant transgenic crops, which are widely cultivated .It also produces antibiotic compounds that are of antifungal activity. Cry proteins are also used as commercial insecticides. Since the genetic diversity and toxic potential of Bt strains differ from region to region, Bt strains have been collected and characterized all over the world from various habitats, including soil, stored-product dusts, insects, deciduous and coniferous sources.

Bacillus thuringiensis var. israelensis (Bti):

Bacillus thuringiensis israelensis (Serotype H-14) is a subspecies of the common insecticidal bacterium Bt. Bti-based products is one of the most efficient and the safest methods to control some larval mosquitoes, black flies' midges' populations. Their very specific and target-oriented mode of action of Bti makes it very safe for human health and non-target organisms.

OCCURRENCE OF Bt:

The occurrence of bacillus thuringiensis is identified in the following natural sources:

Bt TOXIN AND THEIR CLASSIFICATION:

A class of crystalline pore forming proteins produced by strains of Bacillus thuringiensis, and engineered into crop plants to give resistance against insect pests. Their mechanism involves the lysis of midgut epithelial cells by inserting into the target cell membrane and forming pores. There are two types of toxins produced from Bt strains:

Cry(crystal) toxins

Cyt(cytolytic) toxins

Domain 1 = responsible for inserting into the gut membrane and creating a pore where ions can pass freely

Domain 2 = responsible for binding to the receptors on the epithelial lining of the midgut

Domain 3 = responsible to protect the endotoxin from cleavage by gut proteases, or may be involved in ion channel formation, receptor binding, and insect specificity

Cry protin structure:

GENETIES OF Bti:

Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid of Bacillus thuringiensis subsp.israelensis:

MODE OF ACTION OF CRY PROTEIN:

Bti bacterium produces a protein crystal which is toxic only to mosquito and black fly larvae during the spore-forming stage of its life cycle. When the insects feed, these microscopic crystals are ingested by insect larvae. The crystals are dissolved thus converted into toxic protein molecules which destroy the walls of the insect's stomach in the alkaline environment of the susceptible insect's digestive system. The insect stops feeding within hours and dies within days.

Figure 3:mode of action of bt toxin from http://web.utk.edu/jurat/

Bt BASED BIOPESTICIDES

According to the United States Environmental Protection Agency (EPA), "biopesticides" are naturally occurring substances (biochemical pesticides) that

Control pests, microorganisms that control pests (microbial pesticides), and pesticidal substances produced by plants containing added genetic material, plant-incorporated protectants.

The strains of Bt characterized so far affect members of three insect orders:

Bt.japonensis and kumamotoensis (Coleoptera)-used on several turf beetle species.

Bt.gallerie (Coleoptera)-used on Japanese beetles.

COMMERCIAL PRODUCTION:

TABLE 4 : Some Bti products for mosquito and black fly control (possibly not all currently available) (modified from Becker and Margalit 1993).

Product

Formulation

Company

Teknar TC

Powder

Novartis (sold by Triology)

Teknar HP-D

Fluid

Novartis (sold by Triology)

Teknar G

Granules

Novartis (sold by Triology)

VectoBac TP

Powder

Abbott Laboratories

VectoBac 12 AS

Fluid

Abbott Laboratories

VectoBac G

Granules

Abbott Laboratories

VectoBac CG

Abbott Laboratories

Bactimos WP

Powder

Abbott Laboratories

Bactimos G

Granules

Abbott Laboratories

Bactimos

Briquettes/pellets

Abbott Laboratories

Bactimos PP

Abbott Laboratories

Cybate (Australian label)

Fluid

Cyanamid

Skeetal FC

Fluid

Entotec/Novo

BMC WP

Powder

Reuter

Duplex

methoprene + Bti

Zoecon - PPM

GMOs and Bt:

A GMO (genetically modified organism) is defined as 'an organism whose genetic map has been modified in a different way from what happens in nature by cross breeding or natural genetic combination' (directive CEE 90/220 and French law 92/654).

Table 5: Various Genetically Modified crops produced by using Bt :

SL.NO.

CROP

GENE/EVENT

1

Potato

RB gene

2

Cabbage

cry1Ac /cry1Ba & cry1ac3

3

Okra

cry1Ac

4

Corn

cry1Ac + cp4epsp4

5

Brinjal

cry1Ac/cry1Aa & cry1Aabc

6

Cauliflower

cry1Ac /cry1Ba & cry1ac3

7

Tomato

unedited NAD9

8

Rice

cry1Ab, cry1C & bar

9

Groundnut

chitinase gene

GMO REGULATIONS IN INDIA

Some of the regulatory guidelines followed for Genetically Modified Organisms in India are:

1998 "Revised Guidelines for Research in Transgenic Plants and Guidelines for Toxicity and Allergenicity Evaluation of Transgenic Seeds, Plants and Plant Parts" (1998 DBT Guidelines).

Guidelines for the conduct of confined field trials of regulated, GE crops, 2008

Standard Operating Procedures for confined field trials 2008

Guidelines and protocols for food and feed safety assessment of GE crops, 2008.

Bt strains show genetic diversity with different toxic potential mostly due to plasmid exchange between strains (Thomas et al. 2001). Hence, each habitat may contain a novel Bt strain awaiting to be discovered which has a toxic effect on a some target insect group. The aim of this study is to isolate, identify, characterize Bt strains from different soil samples, and to quantify and qualify their cry protein content to study the best strain among all these identified strains which can be used as a effective biopesticide against mosquitoes and black flies' midges.Â

OBJECTIVES:

bti(bacillus thuringiensis var israelensis)

Isolation of Bacillus thuringeinsis (bti) from different soils.

10 cultures will be identified with specific medium (verify with std.)

Culturing of Bacillus thuringiensis

Visualizing by gram's staining

protein(delta-endotoxin) purification for best strains

protein(delta-endotoxin) quantification by Lowry's method

Quantification of protein(delta endotoxin) by ELISA

SDS-PAGE for protein(delta endotoxin) identification

MATERIALS AND METHODs:

SAMPLE COLLECTION:

Five different soil samples were collected from:

Rice field - Chengalpet (Rc)

Flower field - Chengalpet(Fc)

Sugarcane field - Chengalpet(Sc)

Rice field - Tambaram(santhoshpuram)(Rt)

Vegetable field - Tambaram(santhoshpuram)(Rt)

ISOLATION FROM SOIL SAMPLE:

PREPARATION OF SOIL SAMPLE STOCK:

0.85% Saline preparation: 850ml of saline was prepared by adding 7.225g of NaCl in 850ml of nano pure water.

MEDIUM PREPARATION:

Erlenmeyer flask - the sloped sides of the flask are ideal for preparing and autoclaving media.

Scale and weighing papers or trays/"boats" for measuring ingredients.

Empty Petrie plates, which should be sterile and packaged in a plastic bag.

Preparing the media

All dry ingredients except the agar was measured and placed into a 2L Erlenmeyer flask.

1000 ml of nano pure water was added and shook/stirred vigorously to get most of the dry material into solution.

Small aliquot of the solution was taken and the pH was checked in a pH-meter and the pH was adjusted to 7-7.4.

Agar was added and continued mixing - the agar will not go into solution at this stage, but it's important that it not form a large clump on the bottom.

The opening of the flask was covered with tin foil and placed it in an autoclavable metal bin with some water in the bottom (~1cm deep).

The media was autoclaved for a minimum of 20' on the liquid cycle. Pressures typically range from 15-20psi.

After autoclaving, gently the flask was swirled while holding it in water-proof oven or heat-proof gloves. This action is necessary to insure even distribution of the agar in the media; else it often remains denser near the bottom.

The media needs to temper before it is poured into plates. Thus the flask was placed on a heat-proof surface and let it cool. Large volumes (1L or more) should be swirled every 10-20 minutes to redistribute the media within the flask.

The plates were got ready in a laminar air hood. All plates was stacked and poured from bottom to top, lifting the lid of sequential plates (and those above it) to pour the media. Wore the oven-safe mitts while pouring.

The bottom half of the plate should be ~1/2 full, approximately 25mL of media per plate. Plates poured singly generally solidified within 1/2 hour at room temperature.

Solidified plate's were stacked right-side up and slide the original bag in which the empty Petrie plates came.

The plates were turned over so the plates now will be facing agar-side up, exactly as how they should be stored.

Labeled these plates with the type of media and date poured

PLATING PROCEDURE:

The samples of dilutions 10^-1, 10^-4, 10^-6 of each soil samples were taken.

0.1 ml of 10^-1 dilution of Rc sample was poured at the centre of one pre poured agar plate (this center placement makes it easier to spread the sample and to keep the sample away from the edge of the plate)

The sample were spread immediately and spread over the surface by rotating the plate or by a rotating bent glass tube on the surface in clock wise direction.

The procedure was repeated for all 10^-1, 10^-4, 10^-6 dilutions of all samples individually on separate pre pour NYSM agar plates.

Totally 15 such plating was done and all the plates were incubated over night at 28 Í¦ C to obtain maximum growth.

STAINING:

PRINCIPLE: The staining technique is based on the difference between the cell wall compositions of different bacteria. Bacterial cell wall may have higher lipid content or the protein content. Also the stains used in Gram staining have different affinity for these components and they bind with them reversibly or irreversibly. Hence Gram positive bacteria bind the stain irreversibly and cannot be decolorized by alcohol also where as Gram negative bacteria bind the stain reversibly and give it away when washed with water and alcohol. Then they take up the secondary stain become pink stained.

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